Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of G(q/11)-coupled receptors, which subsequently signal to TRPC channels in a G protein- and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of beta-arrestin. Agonist-independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT(1) angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT(1) receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that G(q/11)-coupled receptors function as sensors of membrane stretch in VSM cells.     

Synthesis of novel cationic bolaamphiphiles from vernonia oil and their aggregated structures

The present study describes the synthesis of a novel class of vesicle-forming bolaamphiphiles with choline ester head groups. These bolaamphiphiles were derived from vernonia oil, whose main constituent is vernolic acid, a fatty acid with a unique combination of epoxy, carboxy and unsaturated double bonds. A series of bolaamphiphiles containing amido or ester groups within the hydrophobic domain were synthesized from N,N'-alkylenebis (vernolamides) and alpha,omega-alkylene divernolate ester in a two-stage synthesis comprising opening of the epoxy ring with chloroacetic acid, followed by quaternization with N,N-dimethylaminoethyl acetate to form choline ester head groups. The products were characterized by FT-IR, (1)H and (13)C NMR, and ESI-MS. Vesicles prepared from these bolaamphiphiles have the potential to serve as a targeted drug delivery systems with selective decapsulation in the presence of the enzyme acetylcholine esterase, resulting in site-specific release of the drug.     

Allosteric coupling in pyruvate dehydrogenase kinase 2

Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.     

Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood.Here we examined the contribution of the m₂R-I_KACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m₂R-I_KACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m₂R-I_KACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.     

Lipid nanotechnologies for structural studies of membrane‐associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ～20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ～10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909. © 2014 Wiley Periodicals, Inc.     

The Fate of Cooperation during Range Expansions

Species expand their geographical ranges following an environmental change, long range dispersal, or a new adaptation. Range expansions not only bring an ecological change, but also affect the evolution of the expanding species. Although the dynamics of deleterious, neutral, and beneficial mutations have been extensively studied in expanding populations, the fate of alleles under frequency-dependent selection remains largely unexplored. The dynamics of cooperative alleles are particularly interesting because selection can be both frequency and density dependent, resulting in a coupling between population and evolutionary dynamics. This coupling leads to an increase in the frequency of cooperators at the expansion front, and, under certain conditions, the entire front can be taken over by cooperators. Thus, a mixed population wave can split into an expansion wave of only cooperators followed by an invasion wave of defectors. After the splitting, cooperators increase in abundance by expanding into new territories faster than they are invaded by defectors. Our results not only provide an explanation for the maintenance of cooperation but also elucidate the effect of eco-evolutionary feedback on the maintenance of genetic diversity during range expansions. When cooperators do not split away, we find that defectors can spread much faster with cooperators than they would be able to on their own or by invading cooperators. This enhanced rate of expansion in mixed waves could counterbalance the loss of genetic diversity due to the founder effect for mutations under frequency-dependent selection. Although we focus on cooperator-defector interactions, our analysis could also be relevant for other systems described by reaction-diffusion equations.     

ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.